Method of producing tibolone metabolites by fermentation with Rhizopus stolonifer

ABSTRACT

A new method of producing metabolites of tibolone comprising fermenting tibolone with  Rhizopus stolonifer  (ATCC 12938) resulting in the formation of Δ 4 -Tibolone (C 21 H 28 O 2 ), 6β-Hydroxytibolone, and 15β-Hydroxytibolone (C 21 H 28 O 3 ) is reported.

SUMMARY OF INVENTION

Microbial transformation is an effective tool to synthesize steroidaldrugs with potential clinical applications. Such studies are primarilyuseful in the generation of hydroxylated metabolites for drug toxicitystudies. Fungi, bacteria and yeast have been utilized successfully as invitro models to mimic and predict the metabolic fate of drugs and otherxenobiotics in mammalian systems. Previously, many biotransformationalstudies on various 17α-ethynyl steroids had been carried out with fungaland bacterial strains, which produce hydroxylation of at variouspositions in the chemical structure.

Tibolone (Compound 1) is a synthetic steroid that combines estrogenicand progestogenic properties with androgenic property, which mimic theaction of a male sex hormone. The in vivo metabolism of tibolone (1) inhuman had been studied with the reference to its three metabolites,3α-hydroxytibolone, 3β-hydroxytibolone and Δ⁴-tibolone.

In the current study, tibolone (1) was used as a structural probe toidentify its metabolites produced by a microbial model, and to furtherinvestigate the differences between microbial transformation and humanmetabolism. These metabolic studies showed novel hydroxylation atvarious positions (Compounds 4-20), in which only Compound 4 wasreported previously as metabolite in human metabolism. Compound 1 whenincubated with Rhizopus stolonifer, Fusarium lini, Cunninghamellaelegans and Gibberella fujikuroi, resulted in the formation of a libraryof hydroxyl derivatives. These hydroxytibolones have potential forinhibition of α-glucosidase enzyme. α-Glucosidase (EC 3.2.1.20) is atypical exo-type glycosidase that releases α-glucosides from thenonreducing end side of the substrates. Diabetes mellitus is a chronicmetabolic disorder characterized by high blood glucose levels. Tibolone(1) is used effectively for the treatment of menopausal symptoms and inthe prevention of osteoporosis, as a hormone replacement therapy (HRT).The hormone replacement therapy (HRT) effects on glucose metabolism innon-diabetic obese postmenopausal women.

Glucosidase enzymes are involved in several biological processes such asthe intestinal digestion, the biosynthesis of glycoproteins and thelysosomal catabolism of the glycoconjugates (Homonojirimycin isomers andN-alkylated homonojirimycins: structural and conformational basis ofinhibition of glycosidases. Asano N, Nishida M, Kato A, Kizu H, MatsuiK, Shimada Y, Itoh T, Baba M, Watson A A, Nash R J, Lilley P M, Watkin DJ, Fleet G W., J Med Chem. 1998 Jul. 2; 41(14):2565-71). Intestinalα-glucosidases are involved in the final step of the carbohydratedigestion to convert these into monosaccharides which are absorbed fromthe intestine.

As a result of the catalysis produced by α-glucosidase enzyme in thefinal step in the digestive process of carbohydrates, its inhibitors canretard the uptake of dietary carbohydrates and suppress postprandialhyperglycemia, and could be useful to treat diabetic and/or obesepatients [Novel α-glucosidase Inhibitors with a tetrachlorophthalimideSkeleton., S. Sou, S. Mayumi, H. Takahashi, R. Yamasak, S. Kadoya, M.Sodeoka, and Y. Hashimoto, Bioorg. Med. Chem. Lett., 2000, 10, 1081].

The α-glucosidase inhibitors are effective in lowering the insulinrelease, insulin requirement and some can lower plasma lipids. Theacarbose is a very widely prescribed drug in the management of the typeII diabetes and recently a U.S. Pat. No. 6,387,361 to Rosner describesthe use of acarbose in the treatment of obesity. According to thecriteria issued by WHO (World Health Organization) based on a glucosetolerance test, diabetes mellitus and impaired glucose tolerance(hereinafter sometimes referred to as IGT) are distinguished by thefasting blood glucose level and the blood glucose level 2 hours afterglucose loading. Patients with IGT have high blood glucose levelscompared to those of patients with diabetes mellitus, and are reportedto be at increased risk of developing diabetes mellitus andcomplications of arteriosclerotic diseases. In particular, it is knownthat patients with IGT who have blood glucose levels of 170 mg/dl orabove at 2 hours following glucose loading, i.e., patients withhigh-risk IGT, may develop diabetes mellitus at a high rate [DiabetesFrontier, p. 136, 1992]. With regard to voglibose which is anα-glucosidase inhibitor, there are reports of studies on effects ofvoglibose for insulin-resistant IGT and diabetes [Yakuri-to-Chiryo(Japanese Pharmacology & Therapeutics), 24 (5):213 (1996); Metabol. Exp.Clin., 45:731, 1996]. Voglibose (AO-128) is also known to have effectsof lowering blood glucose level and improving glucose tolerance in rats[Yakuri-to-Chiryo (Japanese Pharmacology & Therapeutics), 19 (11):161(1991); Journal of Nutrition Science and Vitaminology, 45 (1): 33(1992)]. On the contrary, it has also been reported that the effect ofvoglibose in improving glucose tolerance could not be verified in human[Rinsho-Seijinbyo, 22 (4): 109 (1992)]. An antibiotic pradimicin Q asα-glucosidase inhibitor is described in the U.S. Pat. No. 5,091,418 toSwada.

In addition, they have also been used as antiobesity drugs, fungistaticcompounds, insect anti-feedant, anti-viral and immune modulators[Glycosidase inhibitors and their chemotherapeutic value, Part 1. elAshry E S, Rashed N, Shobier A H., Pharmazie. 2000 April;55(4):251-620]. The antiviral activity due to inhibition ofα-glucosidase results form abnormal functionality of glycoproteinsbecause of incomplete modification of glycans. Suppression of thisprocess is the basis of antiviral activity [A glucosidase-Inhibitors aspotential broad based antiviral agents, Anand Mehta, Nicole Zitzmann,Pauline M. Rudd, Timothy M. Block, Raymond A. Dwek, Febs Letters 430(1998)17-22] and decrease in growth rate of tumors [Inhibition ofexperimental metastasis by an alpha-glucosidase inhibitor,1,6-epi-cyclophellitol. Atsumi S, Nosaka C, Ochi Y, Iinuma H, Umezawa K.Cancer Res. 1993 Oct. 15; 53(20):4896-9]. The α-glucosidase inhibitorN-(1,3-dihydroxy-2-propyl)valiolamine is described as a promoter ofcalcium absorption in the U.S. Pat. No. 5,036,081.

In the present invention is reported a surprising discovery that wasmade when it was discovered that several metabolites of tibolone (1) orits hyroxyderivates obtained by fermentation with various fungi arepotent inhibitors of alpha-glucosidase enzyme, a property of thesechemicals that has never been reported before in the prior art. Table 1lists are metabolites of tibolone and their activity against alphaglucosidase enzyme.

TABLE 1 Novel metabolites of tibolone and their activity againstalpha-glucosidase enzyme Compound Name Novel Active Compound 1 Tibolone(C₂₁H₂₈O₂) No No Compound 2 3β-hydroxytibolone (C₂₁H₃₀O₂) No No Compound3 3α-hydroxytibolone (C₂₁H₃₀O₂) No No Compound 4 Δ⁴-Tibolone (C₂₁H₂₈O₂)No Yes Compound 5 6β-Hydroxytibolone (C₂₁H₂₈O₃) Yes Yes Compound 615β-Hydroxytibolone (C₂₁H₂₈O₃) Yes No Compound 7 Δ^(1,4)-Tibolone(C₂₁H₂₆O₂) Yes Yes Compound 8 10β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes NoCompound 9 11α,15β-Dihydroxytibolone (C₂₁H₂₈O₄) Yes No Compound 1011α,15β-Dihydroxy-Δ⁵-tibolone (C₂₁H₂₈O₄) Yes No Compound 11 Δ⁵-Tibolone(C₂₁H₂₈O₂) Yes No Compound 12 6β-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes YesCompound 13 6α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 1415α-Hydroxy-Δ⁴-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 156α-Hydroxy-Δ^(1,4)-tibolone (C₂₁H₂₈O₃) Yes Yes Compound 166β-Methoxy-Δ⁴-tibolone (C₂₂H₃₀O₃) Yes No Compound 173β,6β-Dihydroxytibolone (C₂₁H₃₂O₃) Yes Not studied Compound 183α-Hydroxy-Δ⁵-tibolone (C₂₁H₃₂O₂) Yes Not studied Compound 193α,6β-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃) Yes Not studied Compound 203α,11α-Dihydroxy-Δ⁴-tibolone (C₂₁H₃₂O₃) Yes Not studied

Here we are reporting a new class of α-glucosidase inhibitors i.e.,17α-ethynyl steroids along with comparison of the results with thestandard inhibitors of this enzyme and their method of manufacture usingmicrobial fermentation.

DETAILS OF INVENTION Brief Description of Drawings

FIG. 1 shows the stereochemical structure of the 20 compounds reportedin this invention.

FIG. 1

Fermentation of tibolone (1) with Rhizopus stolonifer (ATCC 12938)yielded two new mono-hydroxylated metabolites, Compounds 4 and 5, and aknown metabolite, Compound 6.

The HREIMS of Compound 4 exhibited the molecular ion (M⁺) at m/z328.2171, corresponding to the formula C₂₁H₂₈O₃, which indicated that anew oxygen functionality was introduced into the molecule duringfermentation period. The IR absorptions were attributed to hydroxyl(3381 cm⁻¹) and carbonyl (1705 cm⁻¹) functionalities, respectively. The¹H NMR spectrum, compared with that of the substrate, showed a newsignal of OH-bearing methine proton at δ 4.04, resonating as a doublet(J=4.0 Hz) with its corresponding carbon resonating at δ 65.9 in ¹³C NMRspectrum which was assigned to C-6 on the basis of HMBC correlations ofH-6 (δ 4.04) with C-5 (δ 122.5) and C-10 (δ 128.4). In the ¹H-¹H COSY45° spectrum, the aforementioned methine proton showed correlation withthe C-7 methine proton resonated at δ_(H) 2.0. The stereochemistry ofC-6 hydroxyl group was determined to be axial by the NOESY correlationsbetween H-6 (δ 4.04) and H-19 (δ 0.76). The above spectral dataconcluded that Compound 4 has an —OH group at C-6 position as comparedto compound 1 and was deduced to be a new metabolite.

The HREIMS of Compound 5 showed the M⁺ at m/z 328.2070, indicating anincrement of 16 mass units as compared to compound 1 in accordance toformula C₂₁H₂₈O₃. The ¹H and ¹³C NMR data of 5 revealed the presence ofa new OH-bearing methine group that resonated at δ_(H) 4.06 (m,W_(1/2)˜10.8 Hz) and δ_(C) 65.5 and deduced for C-15 on the basis ofHMBC spectrum correlations, which showed correlation of C-16 protons(δ_(H) 2.24, 1.7) and C-14 methine proton (δ_(H) 1.85) with C-15 (δ_(C)65.5). The stereochemistry of the newly introduced C-15 hydroxyl groupwas deduced as 13 on the basis of NOESY correlations between H-15 (δ_(H)4.06) and H-14 (δ_(H) 1.85) and multiplicity of H-15 signal at δ 4.06(W_(1/2)˜10.8 Hz). From these spectral data, the new compound 5 wasdeduced to7α-methyl-17α-ethynl-15β,17β-dihydroxy-19-norandrost-5(10)-en-3-one.

The incubation of compound 1 with Fusarium lini (ATCC 9593) for 6 daysalso led to the isolation of a UV active Compound 6 exhibiting the M⁺ atm/z 312.2023 in HREIMS spectrum (C₂₁H₂₈O₂). The ¹H NMR spectrum showed asinglet for an olefinic proton at δ 5.82. Its broad-band decoupled ¹³CNMR spectrum showed, in comparison with that of the substrate 1, thedisappearance of one quaternary carbon signal resonating at δ 128.2 forC-10 and appearance of an olefinic methine carbon at δ 126.4 which wasassigned to the C-4 on the basis of HMQC spectrum, indicating themigration of the C-5/C-10 double bond to C-4/C-5. Thus creating anα,β-unsaturation in Compound 6. The axial orientation of C-10 proton wasassigned on the basis of NOESY coupling between H-10 (δ 2.31) and H-8 (δ1.64). The above spectral data supported the structure of a knownCompound 6 as 7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-4-en-3-onepreviously isolated during human metabolism of tibolone.

The incubation of 1 (600 mg) with Cunninghamella elegans (ATCC 10028b)for six days yielded Compounds 7-10 (FIG. 1). The HREIMS of compound 7showed the M⁺ at m/z 310.2004, in accordance with the formula C₂₁H₂₆O₂.Comparison between the ¹H and ¹³C NMR data of compounds 6 and 7,Compound 7 showed the presence of two additional olefinic signals at δ7.12 (dd, J=8.4, 4.7 Hz, H-1) and 6.62 (dd, J=8.4, 2.6 Hz, H-2) in the¹H NMR spectrum with the corresponding carbons resonated at δ 153.4(C-1) and 127.0 (C-2), respectively, in the ¹³C NMR spectrum in compound7. The presence of a double bond between C-1 and C-2 was further deducedfrom ¹H-¹H COSY 45° correlations between H-1 (δ 7.12), H-2 (δ 6.62) andH-10 (δ 2.42). While HMBC experiment showed interactions of H-1 with C-2(δ 127.0) and C-10 (δ 43.0). The final structure of Compound 7 wasdeduced to be7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-1,4-dien-3-one.

The HREIMS of Compound 8 showed the M⁺ at m/z 328.2090, in agreementwith the formula C₂₁H₂₈O₃ indicating an introduction of a new oxygen inthe molecule, probably in the form of a hydroxyl group. However the ¹HNMR spectrum displayed no resonance for OH-bearing methine proton, but¹³C NMR spectrum showed a downfield oxygen-bearing quaternary carbonresonated at δ 70.3, which was assigned to C-10 through its HMBCinteractions with H-1 (δ 2.36, 2.29) and H-4 (δ 5.77). The10β-hydroxylation was deduced by the β-SCS (substituents chemical shift)of −5.1, −5.4 and −6.9 ppm for C-2, C-8 and C-11, respectively, and bythe downfield shifts of C-1 and C-9 (+5.1 and +7.5, respectively) withrespect to the ¹³C NMR chemical shifts in compounds 4 and 8. Thespectral data supported the structure of a new Compound 8 as7α-methyl-17α-ethynl-10β,17β-dihydroxy-19-norandrost-4-en-3-one.

The HREIMS of Compound 9 showed the M⁺ at m/z 344.2212 supporting theformula C₂₁H₂₈O₄, indicated that two oxygen had been incorporated intothe molecule. The ¹H and ¹³C NMR displayed two OH-bearing methine groupsresonating at δ_(H) 3.43 (ddd, J=15.1, 11.1, 5.0 Hz); and 4.10 (m,W_(1/2)˜8.82 Hz) and δ_(C) 66.1 and 65.4, respectively. The ¹H-¹H COSY45° spectrum showed correlations of H-11 (δ 3.43) with H-9 (δ 1.62) andH₂-12 (δ 2.05, 1.51), and of H-15 (δ 4.10) with H-14 (δ 1.80) and H₂-16(δ 2.01, 1.55). Hydroxylations at C-11 and C-15 was further supported byHMBC assignments, which has exhibited correlations of H₂-12 (δ 2.05,1.51) and Me-18 (δ 0.90) with C-11 (δ 66.1), and correlation of H-14 (δ1.80) with δ 65.4 (C-15). The axial orientation of C-11 proton wasdeduced on the basis of NOESY correlation of H-11 (δ 3.43) with Me-18 (δ0.90) and multiplicity of H-11 signal resonating at δ_(H) 3.43 (ddd,J=15.1, 11.1, 5.0 Hz),³ while n-stereochemistry of the newly introducedOH group at C-15 was deduced by the NOESY correlations between H-14 (δ1.80) and H-15 (δ 4.10) and multiplicity of H-15 signal resonating at δ4.10 (W_(1/2)˜8.8 Hz). The β-orientation of C-10 proton was similar tocompound 6 (FIG. 3). According to this spectral data, the structure wasdeduced to be7α-methyl-17α-ethynl-11α,15β,17β-trihydroxy-19-norandrost-5(10)-en-3-one.

The HREIMS of Compound 10 showed the M⁺ at m/z 344.2341, support formulaC₂₁H₂₈O₄, with an increment of 32 a.m.u. The UV spectrum showed a weakabsorption at 202 nm, while IR showed absorptions at 3312 (OH), 1722(C═O) and 1652 (C═C) cm⁻¹. The ¹H NMR spectrum showed an upfield doubletof olefinic methine proton at δ 5.42 (J=4.2 Hz, H-6), which showed COSY45° correlations with H-7 (δ 1.83). Two additional OH-bearing methineprotons resonating at δ 3.40 (ddd, J=15.3, 11.0, 4.57 Hz) and 3.91 (m,W_(1/2)˜9.9 Hz) were unambiguously assigned to H-11 and H-15 through 2DNMR and ¹³C NMR spectra. The stereochemistry of newly introducedhydroxyl group at C-11 was deduced to be a (equatorial) on the basisNOESY correlation between H-11 (δ 3.40) and H-18 (δ 0.94) and largercoupling constants (J=15.3 Hz) of H-11 signal. The β-orientation of theOH group at C-15 was deduced on the basis of NOESY correlation betweenH-14 (δ 1.85) and H-15 (δ 3.91) and multiplicity of H-15 signal,resonating at δ 3.91 (m, W_(1/2)˜9.9 Hz). The axial β-orientation ofC-10 proton was deduced through NOESY cross peaks between H-10 (δ 2.46)and H-8 (δ 1.59). Based on the above mentioned spectral data, thestructure was deduced as7α-methyl-17α-ethynl-11α,15β,17β-trihydroxy-19-norandrost-5-en-3-one.

Tibolone (1) was fermented with Gibberella fujikuroi (ATCC 10704) for 12days yielding six new mono-hydroxylated Compounds 11-16. The HREIMS ofCompound 11 showed the M⁺ at m/z 312.1456 with corresponding formulaC₂₁H₂₈O₂. The ¹H NMR spectrum showed an upfield doublet of olefinicproton at δ 5.33 (J=4.7 Hz) which was assigned to H-6 through its ¹H-¹HCOSY 45° correlation with H-7 (δ 1.85). The ¹³C NMR spectrum showedlow-field methine carbon resonated at δ 123.4. A double bond between C-5and C-6 was deduced through HMBC coupling between H-6 (δ 5.33) and C-7(δ 34.6). According to the spectral data, the structure was deduced as7α-methyl-17α-ethynl-17β-hydroxy-19-norandrost-5-en-3-one.

Compounds 12 and 13 were found to be epimers and differentiated on thebasis of ¹H NMR and NOESY experiments. The ¹H NMR spectrum of Compound12 displayed a doublet at δ 4.05 (J=4.0 Hz), while compound 13 exhibiteda doublet at δ 3.60 (J=6.4 Hz), while ¹³C NMR spectra of both theisomers 12 and 13 showed OH-bearing methine carbons resonating at δ 65.9and 70.0, respectively. The position of the newly introduced hydroxyl atC-6 in both isomers was inferred from the HMBC coupling. The relativeconfiguration in compound 12 of the new hydroxyl group at C-6 wasinferred on the basis of coupling pattern and NOESY correlations betweenH-6 (δ 4.05) and C-19 methyl protons (δ 0.75), while NOESY spectrum ofcompound 13 also displayed correlations between H-6 (δ 3.60) and C-7methine proton (δ 1.98). The above spectral data concluded thatCompounds 12 and 13 have an —OH group at C-6 position with differentorientations.

The molecular formula of Compound 14 was established as C₂₁H₂₈O₃ byHREIMS (m/z 328.2132). The ¹H and ¹³C NMR spectra of compound 14exhibited a OH-bearing methine group resonated at δ_(H) 4.02 (m,W_(1/2)˜14.9 Hz); δ_(C) 65.9 and was unambiguously assigned to C-15 onthe basis of two-dimensional NMR experiments. In the ¹H-¹H COSY 45°spectrum, the aforementioned methine proton showed correlation with theC-14 methine proton resonating at δ_(H) 1.72. This was further supportedby the HMBC spectrum, which exhibited correlations of C-14 proton (δ_(H)1.72) with C-15 (δ_(C) 65.9). The α-relative configuration of OH groupat C-15 was deduced on the basis of multiplicity of H-15 signalresonating at δ_(H) 4.02 (m, W_(1/2)˜14.9 Hz).¹² This spectral data ledto the structure 14 as7α-methyl-17α-ethynl-15β,17β-dihydroxy-19-norandrost-4-en-3-one.

The HREIMS of Compound 15 showed the M⁺ at m/z 326.2231 (C₂₁H₂₈O₃). Twoadditional doublets in the ¹H NMR spectrum for two olefinic protonsappeared at δ 7.08 (dd, J=8.18, 4.2 Hz) and 6.76 (d, J=8.22 Hz) and onenew hydroxymethine proton, appeared at δ 3.81 (d, J=6.4 Hz). The DEPTspectrum of compound 15 showed two olefinic methine carbon signals at δ154.2 and 126.5, corresponding to a double bond between C-1/C-2, asdeduced on the basis of HMBC correlations of H-1 (δ 7.08) with C-2 (δ126.5) and, of H-2 (δ 6.76) with C-1 (δ 154.2) and C-4 (δ 125.7). OneOH-bearing methine carbon resonated at δ 69.8 was assigned to C-6 basedon ³J correlations between H-6 (δ 3.81) and C-4 (δ 125.7). Theα-orientation of newly hydroxyl group at C-6 was deduced to beequatorial on the basis of NOESY correlations between H-6 (δ 3.81) andH-7 (δ 1.95). According to these spectral studies, the structure ofCompound 15 was deduced to be7α-methyl-17α-ethynl-6α,17β-dihydroxy-19-norandrost-1,4-dien-3-one.

The HREIMS of Compound 16 showed the M⁺ at m/z 343.2356 corresponding tothe formula C₂₂H₃₀O₃. The ¹H NMR spectrum of Compound 16 showed thepresence of a methoxy singlet resonating at δ 3.47, while geminalmethoxy protons were resonated at δ 3.93 (d, J=3.9 Hz). The ¹³C NMRspectrum showed a methoxy carbon signal at δ 57.6 and a methoxy-bearingcarbon resonated at δ 70.2. The position of the newly introduced methoxygroup at C-6 was deduced through HMBC interactions of H-6 (δ 3.93) withC-4 (δ 126.7). The n-orientation of the newly introduced OCH₃ group atC-6 was deduced on the basis of NOESY correlations between H-6 (δ 3.93)and C-19 methyl protons (δ 0.77). The above mentioned spectral data ledto conclude that Compound 16 has the structure as7α-methyl-17α-ethynl-6β-methoxy-17β-hydroxy-19-norandrost-4-en-3-one.

Tibolone (1) (1 g) when reduced with NaBH₄ in dichloromethane yieldedreduced products i.e. 3β-hydroxytibolone (2) and 3α-hydroxytibolone (3)in order to check the effect on hydroxylation at various positions inpresence of C-3 hydroxyl group (α and β isomers) was investigated. Thepresence of hydroxyl group at C-3 pronounced the hydroxylation at C-6 asin case of Compounds 17 and 19 and produced better yield of thesemetabolites as compared to tibolone (1) hydroxyl metabolites. The ¹H NMRspectra of both the isomers 2 and 3 showed OH-bearing geminal methineprotons appeared at δ 3.8 (m, W_(1/2)˜21.5 Hz) and 4.04 (m, W_(1/2)˜10.7Hz), which indicated the reduction of the C-3 ketonic group. Theorientation of C-3 methine proton in the both isomers 2 and 3 wasdeduced from the multiplicity of C-3 proton signals resonated at δ 3.8(m, W_(1/2)˜21.5 Hz, H-3α) and 4.04 (m, W_(1/2)˜10.7 Hz, H-3β),respectively.

Incubation of 3β-hydroxytibolone (2) (300 mg) with Cunninghamellaelegans for twelve days yielded one hydroxyl-bearing Compound 17.Compound 17 showed the W at m/z 330.2346 corresponding to the formulaC₂₁H₃₀O₃ by HREIMS. The ¹H NMR spectrum showed a resonance for OH-bearedmethine proton at δ 3.50 (d, J=3.5 Hz), while corresponding carbonresonated at δ 74.2 in ¹³C NMR spectrum. The position of the newlyintroduced hydroxyl group at C-6 was deduced through HMBC interactions.The α (equatorial) orientation of C-6 proton, geminal to the hydroxylgroup, was deduced on the basis of NOESY correlations between H-6 (δ3.50) and C-19 methyl protons (δ 0.73). The structure of Compound 17 wasidentified as7α-methyl-17α-ethynl-3β,6β,17β-trihydroxy-19-norandrost-5(10)-en-3-one.

Fermentation of 3α-hydroxytibolone (3) (400 mg) with Cunninghamellaelegans for twelve days yielded three polar Compounds 18-20. Compound 18showed the M⁺ at m/z 314.2541 corresponding to the formula C₂₁H₃₀O₂ byHREIMS, while the ¹H and ¹³C NMR spectra showed appearance of olefinicproton at 5.38 (d, J=4.5 Hz) with corresponding carbon resonated at δ132.0 as compared to substrate 3 and was assigned to C-6 methine carbon.The β (axial) orientation of C-10 proton was deduced on the basis ofNOESY correlations. From these data, the new compound 18 was identifiedas 7α-methyl-17α-ethynl-3α,17β-dihydroxy-19-norandrost-5-en-3-one.

The HREI-MS of Compound 19 showed the W at m/z 330.2356 corresponding tothe formula C₂₁H₃₀O₃ by HREIMS. The 1-1 NMR spectrum of 19 showed oneadditional OH-bearing methine proton signal at δ 4.13 (br. s), while ¹³CNMR spectrum showed the resonance of corresponding methine carbon at δ74.0 (C-6). The ¹H-¹H COSY 45° spectrum also showed an allylic couplingbetween H-4 and H-6 (δ 4.13). The α (equatorial) orientation of C-6proton, geminal to hydroxyl group, was deduced on the basis NOESYcorrelations between H-6 and Me-19 protons (δ 0.87). The structure ofCompound 19 was identified as7α-methyl-17α-ethynl-3α,6β,17β-trihydroxy-19-norandrost-4-en-3-one.

Compound 20 showed the M⁺ at m/z 330.2256 in HREIMS spectrum inagreement with the formula C₂₁H₃₀O₃. The ¹H and ¹³C NMR spectrum of 20showed an OH-bearing methine group appeared at δ_(H) 3.80 (ddd, J=13.5,9.5, 3.3 Hz) and δ_(C) 66.3 and was established for C-11 throughhomonuclear COSY correlations between H-11 (δ 3.80), H-18 (δ 1.04) andH₂-12 (δ 2.1, 1.64), while HMBC cross-peak correlations revealed theconnectivities between H-11, H-18 (δ 1.04) and C-11 (δ 66.3). The β(axial) orientation of C-11 proton, was geminal to hydroxyl group, wasdeduced on the basis of larger magnitude of coupling constant of H-11signal at δ_(H) 3.82 (ddd, J=13.5, 9.5, 3.3 Hz) and NOESY correlationsbetween H-11 and Me-19 protons (δ 0.88). The structure of Compound 20was proposed as7α-methyl-17α-ethynl-3α,11α,17β-trihydroxy-19-norandrost-4-en-3-one.

Some hydroxy metabolites of tibolone (1) showed significant inhibitoryactivity against enzyme tyrosinas, mainly hydroxylation at C-6, C-11 andC-15 in Compounds 4, 9 and 10 showed pronounced inhibitory activityagainst enzyme tyrosinas, while α, β unsaturated system (C-4/C-5) inCompound 6 also showed good activity. These metabolites also showedsignificant inhibitory activity against enzyme α-Glucosidase enzyme. Thehydroxyl group at C-6 position (β isomer in Compound 4) showed potentα-Glucosidase inhibitory activity, while other metabolites containing α,β unsaturated system (C-4/C-5) as in Compound 6 and α, β unsaturatedsystem bearing hydroxyl group at C-6 (α and β isomers), C-10β and C-15βshowed pronounced inhibitory activities against enzyme α-Glucosidaseenzyme as in Compounds 8, 12 and 13. Other α, β unsaturated systems(C-1/C-2, C-4/C-5) in Compound 7 and hydroxyl bearing moiety in Compound15 also showed significant activity against enzyme α-Glucosidase enzyme.It was concluded that hydroxylation at C-6 position and α, β unsaturatedsystem pronounced inhibitory activity against α-Glucosidase enzyme.

The fermentation of tibolone (1) by fungus yielded thirteen Compounds4-16. While incubation of hydroxytibolones (2 and 3) with Cunninghamellaelegans yielded four polar Compounds 17-20. The main hydroxylationsoccurred in rings B and D, especially at C-6 and C-1503) positions. TheCompounds 4, 5, 9, 10, 12, 13, 14, 15, and 16 were identified as themain metabolites obtained in these fermentations. Compounds 4, 12, 13,and 15 containing an OH-group at C-6 showed pronounced inhibitoryactivity against α-glucosidase enzyme (Table 2).

TABLE 2 α-Glucosidase inhibitory activities of tibolone (1) and itsanalogs 4-16, as compared with the reference inhibitors IC₅₀ ±S.E.M.^(a) Compound (in μM) 1 NA^(b) 4 225.0 ± 0.00 5 NA^(b) 6 343.0 ±0.00 7 227.0 ± 0.02 8 877.0 ± 0.03 9 NA^(b) 10 NA^(b) 11 NA^(b) 12 653.0± 0.02 13 <70.0 14 <70.0 15 340.0 ± 0.02 16 NA^(b) Deoxynojirimycin^(c)425.6 ± 8.14 Acarbose^(c) 780.0 ± 0.28 *Notes: ^(a)S.E.M. is thestandard error of the mean; ^(b)are the inactive compounds and ^(c)arethe standard inhibitors of the enzyme α-Glucosidase. Compounds 17-20were not tested due to insufficient quantities.

EXPERIMENTAL DETAILS General

Melting points were determined on a Yanaco MP-S3 apparatus. UV spectrawere measured on a Shimadzu UV 240 spectrophotometer. IR spectra wererecorded on a JASCO A-302 spectrophotometer in CHCl₃. ¹H— and ¹³C-NMRspectra were recorded on a Bruker Avance AM-400 spectrometer withtetramethylsilane (TMS) as an internal standard. 2D NMR spectra wererecorded on a Bruker Avance AMX 500 NMR spectrometer. Optical rotationswere measured on JASCO DIP-360 digital polarimeter by using 10 cm celltube. Mass spectra (EI and HREI-MS) were measured in an electron impactmode on Varian MAT 12 or MAT 312 spectrometers and ions are given in m/z(%). TLC was performed on a pre-coated silica gel card (E. Merck), spotswere viewed with ultraviolet light at 254 nm for flouresence quenchingspots and at 366 nm for fluorescent spot and stained by spraying with asolution of ceric sulphate in 10% H₂SO₄. For column chromatography,silica gel (E. Merck, 230-400 mesh). Tibolone (1) was extracted fromLivial-Organon using dichloromethane.

Fungi and Culture Conditions

Microbial cultures of the Fusarium lini (ATCC 9593), Rhizopus stolonifer(ATCC 12938), Cunninghamella elegans (ATCC 10028b) and Gibberellafujikuroi were grown on Sabouraud-4% glucose-agar (Merck) at 25° C. andstored at 4° C. Rhizopus stolonifer (ATCC 12938) medium was prepared byadding glucose (100 g), peptone (25 g), KH₂PO₄ (25 g) and yeast extract(15 g) into distilled water (4 L) and pH was maintained at 5.6. Fusariumlini (ATCC 9593) and Cunninghamella elegans (ATCC 10028b) media wereprepared by mixing the following ingredients into distilled H₂O (3.0 L)in each case: glucose (30.0 g), glycerol (30.0 g), peptone (15.0 g),yeast extract (15.0 g), KH₂PO₄ (15.0 g), and NaCl (15.0 g). Gibberellafujikuroi medium was prepared by adding the following ingredients intodistilled H₂O (3.0 L): glucose (80.0 g), KH₂PO₄ (5.0 g), MgSO₄.2H₂O (1.0g), NH₄NO₃ (0.5 g) and Gibberella trace element solution (2 mL). TheGibberella trace element solution was prepared by mixing Co(NO₃)₂.6H₂O(0.01 g), FeSO₄.7H₂O (0.1 g), CuSO₄.5H₂O (0.1 g), ZnSO₄.7H₂O (0.161 g),MnSO₄.4H₂O (0.01 g) and NH₄ molybdate (0.01 g) into distilled water (100mL).

General Fermentation and Extraction Conditions

The fungal media were transferred into 250 mL conical flasks (100 mLeach) and autoclaved at 121° C. Seed flasks were prepared from three-dayold slant and fermentation was allowed for two days on a shaker at 25°C. The remaining flasks were inoculated from seed flasks. After twodays, tibolone (1) was dissolved in acetone and transferred in eachflask (15 mg/0.5 mL) and the flasks were placed on a rotary shaker (128rpm) at 22° C. for fermentation period. The time course study wascarried out after two days and the transformation was analyzed on TLC.The culture media were filtrated and extracted with CH₂Cl₂. The extractwas dried over anhydrous Na₂SO₄, evaporated under reduced pressure andthe brown gummy crude was analyzed by thin layer chromatography.

Fermentation of Tibolone (1) with Rhizopus stolonifer (ATCC 12938)

Compound 1 (500 mg), dissolved in 15 mL acetone and distributed among 40flasks and allowed them for fermentation process. All the media werefiltered after 3 days and extracted with dichloromethane and evaporatedunder reduced pressure to finally yield brown thick crude (0.90 mg), andthe transformed metabolites were isolated by using columnchromatography. Compound 4 (20 mg) was eluted with petroleum ether andEtOAc (60:40), compound 5 (17 mg) with petroleum ether-EtOAc (58:42) andcompound 6 (40 mg) with petroleum ether-EtOAc (55:45).

Δ⁴-Tibolone (4)

Crystalline solid (20.4 mg); mp 206-208° C.; [α]²⁵ _(D)−145 (c 0.21,CHCl₃); UV (MeOH) λ_(max) (log ε) 235 (3.2) nm; IR (CHCl₃) v_(max) 3402,2150, 1687, 1667, 1017 cm⁻¹; ¹H NMR data in CDCl₃, Tables 1; ¹³C NMR(100 MHz, CDCl₃) δ 198.1 (C-3), 126.4 (C-4), 161.4 (C-5), 38.2 (C-10),79.1 (C-17), 87.5 (C-20), 74.3 (C-21); EIMS m/z (rel. int. %) 312 (M⁺,34,), 245 (53), 229 (33), 187 (17), 173 (18), 161 (20), 147 (28), 135(56), 121 (23), 109 (32), 107 (43), 105 (39), 95 (24), 91 (62), 81 (34),79 (59), 67 (58), 55 (100); HREIMS m/z 312.2023 (calculated forC₂₁H₂₈O₂, 312.2089).

6β-Hydroxytibolone (5)

White amorphous solid (8.2 mg); mp 186-188° C.; [α]²⁵ _(D)−17 (c 0.35,CHCl₃); UV (MeOH) λ_(max) (log ε) 204 (3.7) nm; IR (CHCl₃) v_(max) 3381,2150, 1705, 1668, 1043 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and3; EIMS m/z (rel. int. %) 328 (M⁺, 6), 309 (5), 241 (16), 226 (9), 169(14), 149 (23), 138 (28), 121 (28), 109 (23), 107 (100), 97 (20), 93(21), 81 (26), 71 (22), 69 (41), 55 (64); HREIMS m/z 328.2171(calculated for C₂₁H₂₈O₃, 328.2143).

15β-Hydroxytibolone (6)

White solid (7.6 mg); mp 202-205° C.; [α]²⁵ _(D)+16 (c 0.31, CHCl₃); UV(MeOH) λ_(max) (log E) 203.4 (3.4) nm; IR (CHCl₃) v_(max) 3383, 2162,1708, 1663, 1050 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and 3;EIMS m/z (rel. in %) 328 (M⁺, 3), 312 (100), 245 (27), 229 (36), 203(17), 189 (14), 187 (17), 174 (24), 161 (28), 149 (26), 135 (24), 121(25), 96 (38), 81 (23), 69 (21), 67 (24), 55 (59); HREIMS m/z 328.2070(calculated for C₂₁H₂₈O₃, 328.2038).

Fermentation of Tibolone (1) by Fusarium lini (ATCC 9593) andCunninghamella elegans (ATCC 10028b)

Compound 1 (600 mg), dissolved in 18 mL acetone and distributed among 50flasks, was kept for fermentation. Fermentation was continued for 6 daysand then filtrates were extracted with dichloromethane and evaporatedunder reduced pressure to afford brown thick crude (1.02 gm). Columnchromatography technique was used for the separation of Compounds 7-10from Cunninghamella elegans crude while Fusarium lini yielded one majorCompound 6. Compound 7 (6.2 mg) was eluted with petroleum ether-EtOAc(42:58), compound 8 (15.5 mg) with petroleum ether-EtOAc (38:62), 9 (5.2mg) with petroleum ether-EtOAc (40:60), whereas compound 10 (10.2 mg)with petroleum ether-EtOAc (30:70).

Δ^(1,4)-Tibolone (7)

Amorphous solid (6.2 mg); mp 196-200° C.; [α]²⁵ _(D)−172 (c 0.32,CHCl₃); UV (MeOH) λ_(max) (log ε) 241.5 (4.1) nm; IR (CHCl₃) v_(max)3303, 2154, 1691, 1666, 1044 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables1 and 3; EIMS m/z (rel. int. %) 310 (M⁺, 42), 241 (100), 230 (11), 199(17), 187 (27), 161 (22), 149 (34), 145 (17), 119 (18), 109 (14), 91(62), 67 (62), 55 (100); HREIMS m/z 310.2004 (calculated for C₂₁H₂₆O₂,310.2011).

10β-Hydroxy-Δ⁴-tibolone (8)

White powdered solid (15.5 mg); mp 198-201° C.; [α]²⁵ _(D)+12 (c 0.25,CHCl₃); UV (MeOH) λ_(max) (log ε) 239 (2.9) nm; IR (CHCl₃) v_(max) 3345,2149, 1698, 1649, 1018 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and3; EIMS m/z (rel. int. %) 328 (M⁺, 13), 310 (14), 229 (20), 187 (25),171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109 (44), 107 (43), 91(55), 83 (28), 67 (47), 57 (50), 55 (100); HREIMS m/z 328.2090(calculated for C₂₁H₂₈O₃, 328.2123).

11α,15β-Dihydroxytibolone (9)

White powdered solid (5.2 mg); mp 208-210° C.; [α]²⁵ _(D)−81 (c 0.24,CHCl₃); UV (MeOH) λ_(max) (log ε) 203 (3.3) nm; IR (CHCl₃) v_(max) 3342,2142, 1718, 1636, 1028 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables 1 and3; EIMS m/z (rel. int. %) 344 (M⁺, 13), 310 (14), 229 (20), 187 (25),171 (26), 161 (25), 149 (48), 136 (32), 124 (55), 109 (44), 107 (43), 91(55), 83 (28), 67 (47), 57 (50), 55 (100); HREIMS m/z 344.2212(calculated for C₂₁H₂₈O₄, 344.2234).

11α,15β-Dihydroxy-Δ⁵-tibolone (10)

White powdered solid (7.3 mg); mp 207-211° C.; [α]²⁵ _(D)+27 (c 0.28,CHCl₃); UV (MeOH) λ_(max) (log ε) 202.4 (3.4) nm; IR (CHCl₃) v_(max)3312, 2102, 1722, 1652, 1057 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, Tables1 and 3; EIMS m/z (rel. int. %) 344 (M⁺, 8), 310 (14), 229 (20), 187(25), 171 (26), 161 (54), 149 (87), 136 (32), 124 (55), 109 (44), 107(43), 91 (55), 83 (28), 67 (74), 57 (50), 55 (100); HREIMS m/z 344.2341(calculated for C₂₁H₂₈O₄, 344.2316).

Fermentation of Tibolone (1) by Gibberella fujikuroi (ATCC 10704)

Compound 1 (850 mg) was dissolved in 20 mL acetone and distributed among30 flasks for fermentation for 12 days. After fermentation, media wasextracted with dichloromethane and evaporated to get a crude extract(1.22 gm). Column chromatography technique was used for the separationof Compounds 11-16 from crude extract. Compound 11 (5.2 mg) was elutedwith petroleum ether-EtOAc (54:46), compound 12 (10.2 mg) with petroleumether-EtOAc (50:50), compound 13 (11.2 mg) with petroleum ether-EtOAc(45:55), compound 14 (8.3 mg) with petroleum ether-EtOAc (38:58),compound 15 (9.5 mg) with petroleum ether-EtOAc (30:70) and compound 16(10.3 mg) with petroleum ether-EtOAc (35:75).

Δ⁵-Tibolone (11)

White powdered solid (5.6 mg); mp 201-205° C.; [α]²⁵ _(D)−17 (c 0.20,CHCl₃); UV (MeOH) λ_(max) (log ε) 203.6 (3.9) nm; IR (CHCl₃) v_(max)3341, 2157, 1728, 1642, 1088 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMSm/z (rel. int. %) 312 (M⁺, 42), 242 (100), 227 (63), 187 (27), 161 (22),149 (34), 124 (26), 91 (21), 67 (23), 55 (100); HREIMS m/z 312.1456(calculated for C₂₁H₂₈O₂, 312.1513).

6β-Hydroxy-Δ⁴-tibolone (12)

White powdered solid (9.0 mg); mp 189-93° C.; [α]²⁵ _(D)−101 (c 0.41,CHCl₃); UV

(MeOH) λ_(max) (log ε) 239.5 (2.1) nm; IR (CHCl₃) v_(max) 3332, 2128,1698, 1651, 1063 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃, and 3; EIMS m/z(rel. int. %) 328 (M⁺, 13), 312 (13), 245 (17), 229 (34), 189 (17), 187(17), 161 (28), 149 (49), 121 (45), 91 (38), 69 (35), 67 (36), 55 (100);HREIMS m/z 328.2176 (calculated for C₂₁H₂₈O₃, 328.2116).

6α-Hydroxy-Δ⁴-tibolone (13)

White powdered solid (7.0 mg); mp 187-190° C.; [α]²⁵ _(D)+66.2 (c 0.35,CHCl₃); UV (MeOH) λ_(max) (log ε) 240.1 (3.7) nm; IR (CHCl₃) v_(max)3342, 2106, 1683, 1659, 1061 cm⁻¹; ¹H and ¹³C NMR data in CDCl; EIMS m/z(rel. in %) 328 (M⁺, 56), 312 (11), 245 (45), 229 (6), 201 (17), 187(14), 171 (20), 161 (34), 149 (57), 135 (24), 121 (46), 91 (34), 81(34), 67 (4), 55 (59); HREIMS m/z 328.2132 (calculated for C₂₁H₂₈O₃,328.2205).

15α-Hydroxy-Δ⁴-tibolone (14)

White powdered solid (8.3 mg); mp 203-205° C.; [α]²⁵ _(D)−21.3 (c 0.34,CHCl₃); UV (MeOH) λ_(max) (log ε) 237.6 (2.6) nm; IR (CHCl₃) v_(max)3401, 2176, 1691, 1662, 1060 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMSm/z (rel. int. %) 328 (M⁺, 3), 312 (100), 245 (27), 229 (36), 203 (17),189 (10), 187 (17), 174 (14), 161 (56), 149 (26), 135 (5), 121 (67), 96(38), 81 (23), 69 (15), 67 (34), 55 (51); HREIMS m/z 328.2212(calculated for C₂₁H₂₈O₃, 328.2283).

6α-Hydroxy-Δ^(1,4)-tibolone (15)

White powdered solid (5.5 mg); mp 193-196° C.; [α]²⁵ _(D)+44 (c 0.25,CHCl₃); UV (MeOH) λ_(max) (log ε) 244 (3.7) nm; IR (CHCl₃) v_(max) 3441,2123, 1680, 1637, 1045 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z(rel. in %) 326 (M⁺, 13), 312 (34), 245 (27), 229 (67), 203 (5), 189(14), 187 (14), 174 (24), 161 (56), 149 (26), 135 (24), 121 (100), 96(45), 81 (23), 69 (12), 67 (24), 55 (34); HREIMS m/z 326.2231(calculated for C₂₁H₂₈O₃, 326.2292).

6β-Methoxy-Δ⁴-tibolone (16)

White powdered solid (8.0 mg); mp 206-209° C.; [α]²⁵ _(D)−18 (c 0.42,CHCl₃,); UV (MeOH) λ_(max) (log ε) 242.1 (3.1) nm; IR (CHCl₃) v_(max)3323, 2153, 1691, 1661, 1101 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMSm/z (rel. in %) 343 (M⁺, 6), 312 (17), 245 (27), 229 (32), 203 (17), 189(14), 187 (17), 174 (25), 161 (28), 149 (26), 135 (24), 121 (25),91(100), 81 (23), 69 (21), 67 (23), 55 (45); HREIMS m/z 343.2356(calculated for C₂₂H₃₀O₃, 343.2338).

Fermentation of 3β-Hydroxytibolone (2) by Cunninghamella elegans (ATCC10028b)

Compound 1 (1 g) was dissolved in dry dichloromethane (100 mL) andcooled in an ice bath to 0° C. Sodium borohydride (500 mg) was added tothe solution in portions whilst stirring. The mixture was stirred atroom temperature for five hours. Acetic acid (50 mL) was destroyed theexcess Sodium borohydride and the dichloromethane was removed in vacuo.Water (100 mL) was added to the resulting oily product, and thesuspension was extracted with ethyl acetate (1 L). The ethyl acetateextract was washed with sodium hydrogen carbonate solution (300 mL),water and brine. The removal of the solvent on a rotary evaporatorafforded crude extract (1.2 g). Column chromatography technique was usedfor the separation of these hydroxytibolones, compounds 2 and 3.

Compound 2 (300 mg) was eluted with petroleum ether-EtOAc (50:50) andcompound 3 (400 mg) was eluted with petroleum ether-EtOAc (48:52).Compound 2 (300 mg), dissolved in 15 mL acetone and distributed among 40flasks, was kept for fermentation. Fermentation was continued for 12days and then filtrates were extracted with CH₂Cl₂ and evaporated underreduced pressure to afford brown thick crude (0.81 gm). Columnchromatography technique was used for the separation of one hydroxylCompound 17 from crude extract. Compound 17 (6.2 mg) was eluted withpetroleum ether-EtOAc (40:60).

3β,6β-Dihydroxytibolone (17)

White powdered solid (4.2 mg); mp 206-209° C.; [α]²⁵ _(D)+105 (c 0.31,CHCl₃); UV (MeOH) λ_(max) (log ε) 201 (3.1) nm; IR (CHCl₃) v_(max) 3353,2164, 1663, 1078 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z (rel. in%) 330 (M⁺, 7), 312 (17), 245 (27), 229 (32), 203 (17), 189 (14), 187(17), 174 (25), 161 (28), 149 (26), 135 (24), 121 (25), 91 (100), 81(23), 69 (21), 67 (23), 55 (45); HREIMS m/z 330.2346 (calculated forC₂₁H₃₂O₃, 328.2374).

Fermentation of 3α-Hydroxytibolone (3) by Cunninghamella elegans (ATCC10028b) Compound 3 (400 mg), dissolved in 18 mL acetone and distributedamong 50 flasks, was kept for fermentation. Fermentation was continuedfor 12 days and then filtrates were extracted with dichloromethane andevaporated under reduced pressure to afford brown thick crude (0.89 gm).Column chromatography technique was used for the separation of hydroxyCompounds 18-20 from crude extract. Compound 18 (11.3 mg) was elutedwith petroleum ether-EtOAc (40:60). Compound 19 (5.2 mg) was eluted withpetroleum ether-EtOAc (54:46) and compound 20 (10.2 mg) with petroleumether-EtOAc (50:50).

3α-Hydroxy-Δ⁵-tibolone (18)

White powdered solid (4.6 mg); mp 206-207° C.; [α]²⁵ _(D)+52.2 (c 0.23,CHCl₃); UV (MeOH) λ_(max) (log ε) 204.2 (4.0) nm; IR (CHCl₃) v_(max)3313, 2154, 1646, 1038 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z(rel. in %) 314 (M⁺, 8), 312 (17), 245 (27), 229 (32), 203 (17), 189(14), 187 (17), 174 (25), 161 (28), 149 (21), 135 (24), 121 (23), 91(11), 81 (23), 69 (21), 67 (21), 55 (45); HREIMS: m/z 314.2541(calculated for C₂₁H₃₂O₂, 314.2597).

3α,6β-Dihydroxy-Δ⁴-tibolone (19)

White powdered solid (4.2 mg); mp 201-205° C.; [α]²⁵ _(D)−18 (c 0.31,CHCl₃); UV (MeOH) λ_(max) (log ε) 201.2 (2.9) nm; IR (CHCl₃) v_(max)3323, 2156, 1665, 1118 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z(rel. in %) 330 (M⁺, 6), 312 (17), 245 (27), 229 (32), 203 (17), 189(14), 187 (14), 174 (25), 161 (21), 149 (26), 135 (22), 121 (25), 91(100), 81 (23), 69 (21), 67 (23), 55 (45); HREIMS m/z 330.2356(calculated for C₂₁H₃₂O₃, 330.2338).

3α,11α-Dihydroxy-Δ⁴-tibolone (20)

White powdered solid (5.1 mg); mp 199-201° C.; [α]²⁵ _(D)+61 (c 0.45,CHCl₃); UV (MeOH) λ_(max) (log ε) 202.6 (3.5) nm; IR (CHCl₃) v_(max)3323, 2143, 1668, 1105 cm⁻¹; ¹H and ¹³C NMR data in CDCl₃; EIMS m/z(rel. int. %) 330 (M⁺, 4), 312 (14), 245 (27), 229 (42), 203 (17), 189(34), 187 (17), 174 (19), 161 (28), 149 (26), 135 (35), 121 (25), 91(98), 81 (23), 69 (67), 67 (23), 55 (51); HREIMS m/z 330.2256(calculated for C₂₁H₃₂O₃, 330.2238).

α-Glucosidase Enzyme Inhibition Assay.

α-Glucosidase (E.C.3.2.1.20) enzyme inhibition assay has been performedaccording to the slightly modified method of Matsui et al. α-Glucosidase(E.C.3.2.1.20) from Saccharomyces sp. was purchased from Wako PureChemical Industries Ltd. (Wako 076-02841). The inhibition has beenmeasured spectrophotometrically at pH 6.9 and at 37° C. using 0.7 mMp-nitrophenyl α-D glucopyranoside (PNP-G) as a substrate and 250 munits/mL enzyme in 50 mM sodium phosphate buffer containing 100 mM NaCl.1-Deoxynojirimycin (0.425 mM) and acarbose (0.78 Mm) were used aspositive controls. The increment in absorption at 400 nm due to thehydrolysis of PNP-G by α-glucosidase was monitored continuously with thespectrophotometer (Molecular Devices, USA). The concentrations of thetest compounds, which inhibited the hydrolysis of PNP-G by α-glucosidaseby 50% (IC₅₀), were determined by monitoring the effect of increasingthe concentration of these compounds on the inhibition values. The IC₅₀values were then calculated using EZ-Fit enzyme kinetics program(Perrella Scientific Inc., Amherst, Mass., U.S.A.).

REFERENCES

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1. A method of producing 6β-Hydroxytibolone, 15β-Hydroxytibolone, andΔ4-Tibolone by contacting tibolone with Rhizopus stolonifer (TSY 0471)under suitable fermentation conditions.